In very recent years, methods have been developed (see "Molecular Cloning of Recombinant DNA", eds., W. A. Scott and R. Werner, Academic Press Inc., 1977)
(1) for the in vitro joining by DNA ligase of a DNA segment to be cloned (scheme 1, structure I) to a cloning vehicle (DNA capable of independent replication, structure II), PA0 (2) for introducing the hybrid DNA molecule (recombinant DNA, structure III) into a suitable cell, PA0 (3) for selecting and identifying the transformed cells carrying the desired hybrid DNA (cloned DNA as a hybrid DNA, structure IV), PA0 (4) for amplifying the desired cloned DNA in the transformed cells, and PA0 (5) for expressing the cloned DNA as a protein product. In most reported cases, DNA molecules isolated from cells or viruses have been fragmented by restriction enzyme digestion or by physical shearing before cloning. ##STR1## Protein synthesis in bacteria at a site located within a transferred DNA segment derived from mouse was shown by Chang et al. (Cell 6, 231-244, 1975). Still other examples of the cloning of natural foreign DNA have been described recently.
Methods for the total chemical synthesis of oligodeoxynucleotides of up to 20 nucleotides long, have been well established by using either the phosphodiester method (Khorana, H. G., J. Mol. Biol. 72, 209, 1972) or the improved phosphotriester method (Hsiung, H. M., and Narang, S. A., Nucleic Acids Res. 6, 1371, 1979; Narang, S. A. et al., Methods in Enzymology, Vol. 65, 610, 1979, and Vol. 68, 90, 1979). The latter method is now the preferred method because of its higher speed, better yield and purity of products, and has been used to prepare defined DNA sequences of longer length.
A few chemically-synthesized DNA sequences, such as the lactose operator (Marians, Wu et al., Nature 263, 744, 1976) and the tyrosine tRNA gene (Khorana, Science 203, 614, 1979), have been successfully cloned in E. coli and the expression of the cloned DNA detected in subsequent cultures. Recent reports have indicated that human brain hormone somatostatin (14 amino acids) has been produced in a transformed bacterial host which had the transferred synthesized gene (Itakura et al., Science 198, 1056, 1977). More recently, reports have appeared that human growth hormone has been produced in bacteria transformed with transferred genetic material comprising the appropriate gene. This latter gene was prepared by copying to DNA part from isolated mRNA (from human pituitary) and synthesizing the remaining part, then joining the two (Goedell et al., Nature 281, 544, 1979).
In the pancreas of animals, preproinsulin (Chan, S. J. and Steiner, D. F., Proc. Nat. Acad. Sci. 73, 1964, 1976) is synthesized as the precursor of insulin. The general structure of proinsulin (Formula 1) is NH.sub.2 -B chain-(C peptide)-A chain-COOH; it is converted to insulin by the action of peptidases in the pancreatic islet tissue which removed the C peptide by cleavage at the positions of the two arrows shown in Formula 1. The B-chain and A-chain of insulin are held together by two disulfide cross-linkages.
Using a biological method, Ullrich et al., (Science 196, 1313, 1977) and Villa-Komoroff, et al., (Proc. Nat. Acad. Sci. 75, 3727, 1978) succeeded in cloning the coding region of rat proinsulin I. However, the cloned gene included extraneous sequences and production of biologically active insulin was not achieved. Using a chemical method, Crea et al. (Proc. Nat. Acad. Sci. 75, 5765, 1978) synthesized, and Goeddel et al. (Proc. Nat. Acad. Sci. 76, 106, 1979) cloned, an insulin A-chain gene and a B-chain gene. The codons selected for these synthetic genes were arbitrary and quite different from the natural human DNA sequence. The construction of these genes was tedious. On culturing, the bacteria produced an insulin A-chain protein and B-chain protein which were separately treated to remove the extraneous .beta.-galactosidase and methionine. The production of A-chain and B-chain proteins was rather poor.
In U.S. patent application Ser. No. 843,422, filed Oct. 19, 1977, by R. Wu, C. P. Bahl and S. A. Narang, adaptor molecules were described for attachment to the ends of DNA sequences for joining to cloning vehicles or other DNA. These adaptors comprise DNA (oligonucleotide) sequences having particular nucleotide segments which are recognition sites for restriction endonucleases. These adaptors can be used to provide an enzyme recognition site on a duplex DNA sequence or to change from one type of site to another. ##STR2##